Plasmids found in nature are distributed to dividing cells so that daughter cells receive at least one copy. Bacterial plasmid systems utilize varying strategies to ensure correct partitioning of plasmids to progeny cells. Maintaining plasmids at high copy number increases the probability that daughter cells inherit at least one plasmid by random distribution so that the probability of progeny cells inheriting no plasmids is extremely low. Although random distribution systems theoretically can function to partition plasmids to dividing cells, plasmids which are maintained at unit or low copy number per cell must rely on active partition mechanisms. For example, plasmids which are maintained at low copy or one copy per cell such as the E. coli F factor (incF1), plasmid prophage P1(incY) and R1 and NR1 (incF2), must utilize active partitioning systems to maintain plasmids in the dividing cell population, since random distribution of plasmid would predict a loss rate of 25% per generation. Further, some higher copy number plasmids rely on site specific recombinases to resolve multimer formation, which functionally reduce the copy number of plasmids in cells and their ability to randomly partition by free diffusion.
Several active partitioning systems have been described for several families of bacterial plasmids. These systems share some common features. Plasmid replication functions can usually be assigned to distinct regions of plasmids. Plasmid replication regions (rep) of, for example P1 or F, can be independently maintained in cells. However, miniF or miniP1 plasmids lacking partition regions are unstable and are lost from the population at frequencies predicted from random distribution.
The development of plasmid vectors for the bacterial expression of heterologous genes for commercial purposes has been extensively documented. Numerous cloning vehicles based on various plasmid replicons have been described and used for production of proteins in E. coli and other bacteria. In the development of cloning vehicles suitable for expression of foreign proteins, the usual strategy has been to design plasmids of low molecular weight with high or regulated copy number. These strategies have sometimes led to the elimination of partition functions which would otherwise lead to stable plasmid inheritance. High copy number cloning vectors for the construction of production organisms often show segregational instability.
The most common strategy for obtaining stable plasmid replication and inheritance in the host population in fermentation has been the inclusion of drug-resistance determinants on the cloning vehicle. Although addition of drugs to growth medium allows selection for cells containing plasmid, addition of antibiotics is unacceptable in many instances because of cost and possible contamination of the end product.
Several alternate strategies have been developed to achieve stable plasmid maintenance during fermentation. For example, the parB locus of R1 has been subcloned into a variety of plasmid common cloning vectors. Resulting plasmids containing the R1 parB region showed enhanced stability when cultured in the absence of selective pressure. Likewise, the sop region of F can stabilize unstable oriC plasmids and P1 par can stabilize a mini-F plasmid that lacks its own partition functions. The stability of cloning vectors containing tryptophan operon genes was increased by addition of the par locus of pSC101 and the unstable multicopy vector derived from p15a (pACYC184) was stabilized by the pSC101 par locus. Other partition regions, such as a partition region from a Salmonella typhimurium virulence plasmid, have also been used successfully to stabilize cloning vehicles.
Although cloning vehicles can essentially be stabilized by the addition of partition regions from stable plasmids, drug resistance determinants are still commonly used during fermentation. Drug-resistance markers are convenient for introduction of plasmids into host bacterial cells. Alternatively, plasmids can be introduced into recipient bacterial cells by complementation of host chromosomal mutations by plasmid-borne genes. Complementation systems rely on the construction of particular host chromosomal mutations, but can be used reliably to circumvent the inclusion of antibiotics to the culture medium. For example, complementation of nutritional defects can lead to plasmid stabilization. Complementation of a chromosomal mutation for aspartic semialdehyde dehydrogenase (asd) in Salmonella typhimurium or D-alanine racemase mutation (dal) in Bacillus subtilis, which each lead to faulty cell wall biosynthesis and cell lysis, yields stable plasmid inheritance in the absence of selection in all viable cells of the culture. Both the asd and dal mutations can be phenotypically repaired by supplementation with nutritional additives. On the other hand, complementation of an essential gene of the host, which defect cannot be overcome by nutritional supplementation can also stabilize plasmids. For example, an E. coli gene, ssb, is required for DNA replication and cell viability and prevents the accumulation of plasmidless cells during fermentation in a bioreactor when incorporated into a plasmid and can be used to complement a chromosomal ssb defect. Analogously, a plasmid borne copy of valyl tRNA synthetase stabilizes plasmids in E. coli containing a chromosomal temperature-sensitive valyl tRNA synthetase. Plasmids can also be stabilized by inclusion of a bacteriophage repressor gene, the loss of which leads to induction of host prophage and cell death.